Double-stranded cDNA fragments were synthesized from wild type HAV strain HM-175 RNA. Cloned cDNA was used as a probe for detecting HAV RNA in tissue culture, serum, and fecal specimens by hybridization. Hybridization experiments also demonstrated that probes taken from any region of the HAV genome will not hybridize to RNA or cloned cDNA from a variety of other picornaviruses. In addition, from analysis of the complete nucleotide and predicted amino acid sequences of this genome and comparison with sequences from other picornaviruses, we have concluded that HAV has typical picornaviral genome organization but widely divergent sequences and it should be classified separately from other picornaviral genera. HAV VPg was demonstrated by the interaction between antibodies directed against a synthetic peptide, representing the VPg amino acid sequence predicted from cloned cDNA, and a covalent HAV RNA-protein (VPg) complex. The baculovirus expression system is being used in attempts to express the structural and nonstructural proteins of HAV.